BIOCHEM CHAP 3 - Protein Sequencing

Primary structure of protein

linear sequence of amino acids & location of any disulfide bridges

Fredrick Sanger

first protein sequence - of bovine insulin

3 steps of protein primary structure study

1. Separate into individual polypeptide chains (cleave disulfide bonds)

2. on each chain - end terminus analysis (amino acids on N & C)

3. sequencing (can be done without step 2)

How to break disulfide bonds *****

1. oxidizing with performic acid - changes cysteine to cystic acid (DRAW!)

2. a) reduce with XS thiol to reduce disulfide bonds (oxygen can make it reoxidize)

b) 2nd “Cap” thiols with iodoacetic acid - gives thioether

Does insulin have subunits

no - does not have quaternary structure

End terminus analysis

look at what amino acids are present at each terminus

C-terminus analysis

uses carboxypeptidases to cleave protein peptide bonds from end inwards - determine end terminus

Carboxypeptidases

are exopeptidases (exoproteases) that cleaves AA’s at the C-terminus (quick so only one is released)

Types of Carboxypeptidases

B - cut after +1 basic side chains (lysine & arginine)

A - cut after “all the others:

Reagents that react with NH2-terminus of polypeptide

• flurodinitrobenzene

• dabsyl chloride

• dansyl chloride

Dansyl/Dabsyl chloride reaction

• need weak base conditions (NH20) - makes it nucleophillic

• attacks chloride on dansyl chloride - HCL released

• N - terminus attached & labeled

How to determine what N-terminus is after dansyl chloride

• Add strong acid for acid-catalyzed hydrolysis - breaks all peptide bonds

• only the N-terminus is labeled & is flourescent

Lysine test results w/ N-terminus analysis

Lysine will give false positive - because it reacts with reagents (NH2 side chains)

Flurodinitrobenzene (DNFB) N-Terminus reactions

undergoes NAS (weak base conditions - so nucloephillic)

aromatic needs EWG (Flourine)

lysine gives false positive

is DNFB a sequencing reagent

NO! only a detecting reageant

PITC / Edward’s Reagent

1. PITC attaches via Nu attack

2. anhydrous triflouroacetic acid (TFA) for hydrolysis - promotes self cleaving

3. cleaves off N-terminal residue

4. rearranges with mild acid - N-phenyl group switches

left with PTH amino acid - can extract with organic solvent

Edward’s Reagent differentiation

does not need mild acid - do not need to break peptide bonds

can break in multiple cycles - so you can use for SEQUENCING!! (is automated into a protein sequencer - very efficient)

efficiency of Edward’s reagant

98% efficient - 2% can cumulate so can only sequence 100 amino acids (small protein)

Fragmentation Methods

1. endoproteases/endopeptidases (cut protein sites in the middle of protein chain)

   1. eg - trypsin/chymotrypsin/elastase

2. w/ Cynaogen Bromide (CNBr)

Trypsin

cuts only after lysine & arginine

Chymotrypsin

cuts after bulky benzene containing side chains (Phe, Trp, Tyr)

Elastase

cuts after small neutral residues (Ala, Gly, Ser, Val - NOT PROLINE)

CNBr

cleaves on carboxyl side of methionine (Cyanogen Bromide)

Why are fragmentation methods needed

because whole proteins are too large and complex to sequence or measure directly

How to determine where disulfide bonds occur

• reduce bond & separate the chains

• break them into smaller peptides via tryptic digest/ mix of another technique

• repeat without breaking disulfide bonds

• overlap data to get bigger picture

DNA sequencing relation to protein sequencing

many protein sequences are known from gene (mRNA) sequencing - INDIRECT SEQUENCING

Mass Spectrometry

3rd method to determine protein sequencing

Peptide Mass Fingerprinting

• use trypsin digestion to determine protein sequence - uses MALDI-TOF & ESI

MALDI TOF & ESI

gentle vaporization techniques to get protein fragments into gas phase

• MALDI TOF (uses laser to pass on energy of protein)